Prevalence and properties of mecC methicillin-resistant Staphylococcus aureus (MRSA) in bovine bulk tank milk in Great Britain.

OBJECTIVES: mecC methicillin-resistant Staphylococcus aureus (MRSA) represent a newly recognized form of MRSA, distinguished by the possession of a divergent mecA homologue, mecC. The first isolate to be identified came from bovine milk, but there are few data on the prevalence of mecC MRSA among dairy cattle. The aim of this study was to conduct a prevalence study of mecC MRSA among dairy farms in Great Britain. METHODS: Test farms were randomly selected by random order generation and bulk tank samples were tested for the presence of mecC MRSA by broth enrichment and plating onto chromogenic agar. All MRSA isolated were screened by PCR for mecA and mecC, and mecC MRSA were further characterized by multilocus sequence typing, spa typing and antimicrobial susceptibility testing. RESULTS: mecC MRSA were detected on 10 of 465 dairy farms sampled in England and Wales (prevalence 2.15%, 95% CI 1.17%-3.91%), but not from 625 farms sampled in Scotland (95% CI of prevalence 0%-0.61%). Seven isolates belonged to sequence type (ST) 425, while the other three belonged to clonal complex 130. Resistance to non-ß-lactam antibiotics was uncommon. All 10 isolates produced a negative result by slide agglutination for penicillin-binding protein 2a. mecA MRSA ST398 was detected on one farm in England. CONCLUSIONS: mecC MRSA is widely distributed among dairy farms in Great Britain, but this distribution is not uniform across the whole country. These results provide an important baseline dataset to monitor the epidemiology of this emerging form of MRSA.

Prevalence and characterization of human mecC methicillin-resistant Staphylococcus aureus isolates in England.

OBJECTIVES: There are limited data available on the epidemiology and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the human population that encode the recently described mecA homologue, mecC. To address this knowledge gap we undertook a prospective prevalence study in England to determine the prevalence of mecC among MRSA isolates. PATIENTS AND METHODS: Three hundred and thirty-five sequential MRSA isolates from individual patients were collected from each of six clinical microbiology laboratories in England during 2011-12. These were tested by PCR or genome sequencing to differentiate those encoding mecA and mecC. mecC-positive isolates were further characterized by multilocus sequence typing, spa typing, antimicrobial susceptibility profile and detection of PBP2a using commercially available kits. RESULTS: Nine out of the 2010 MRSA isolates tested were mecC positive, indicating a prevalence among MRSA in England of 0.45% (95% CI 0.24%-0.85%). The remainder were mecA positive. Eight out of these nine mecC MRSA isolates belonged to clonal complex 130, the other being sequence type 425. Resistance to non-ß-lactam antibiotics was rare among these mecC MRSA isolates and all were phenotypically identified as MRSA using oxacillin and cefoxitin according to BSAC disc diffusion methodology. However, all nine mecC isolates gave a negative result using three different commercial PBP2a detection assays. CONCLUSIONS: mecC MRSA are currently rare among MRSA isolated from humans in England and this study provides an important baseline prevalence rate to monitor future changes, which may be important given the increasing prevalence of mecC MRSA reported in Denmark.

First detection of livestock-associated meticillin-resistant Staphylococcus aureus CC398 in bulk tank milk in the United Kingdom, January to July 2012.

Livestock-associated meticillin-resistant Staphylococcus aureus belonging to clonal complex 398 (LA-MRSA CC398) is an important cause of zoonotic infections in several countries, but there is only a single published report of this lineage from the United Kingdom (UK). Here, we describe the isolation of LA-MRSA CC398 from bulk tank milk from five geographically dispersed farms in the UK. Our findings suggest that LA-MRSA CC398 is established in livestock in the UK. Awareness of the potential occupational risks and surveillance in other food-producing animal species should be promoted.

Development of an assay for in vivo human neutrophil elastase activity. Increased elastase activity in patients with alpha 1-proteinase inhibitor deficiency.

Leukocyte extracts contain enzymes that digest fibrinogen and release a fibrinopeptide A-containing fragment. This study was undertaken to identify the responsible proteinase and to characterize the fibrinopeptide A-containing fragment so that it could be used as an index of enzyme activity. Both the fibrinogenolytic activity and the release of the fibrinopeptide A-containing fragment mediated by the leukocyte extracts were shown to be due to human neutrophil elastase (HNE) by the following criteria: activity was completely blocked by a specific HNE inhibitor or by adsorbing HNE from the extracts with a monospecific antibody and reconstitution with purified HNE restored the ability to release the fibrinopeptide A-containing fragment. This fragment was not released by a variety of other proteinases or by HNE-inhibitor complexes indicating that, at least with respect to the enzymes tested, it is a specific product of HNE and its release requires the free enzyme. By separating the products of HNE digestion of fibrinogen using high performance liquid chromatography, identifying the immunoreactive fractions and subjecting them to amino acid analysis, the fragment was identified as A alpha 1-21, indicating an HNE cleavage site at the Val(A alpha 21)-Glu(A alpha 22) bond. The mean plasma A alpha 1-21 level was markedly higher in patients with alpha 1-proteinase inhibitor deficiency as compared to healthy controls (0.2 nM vs. 7.9 nM; P less than 0.0001), consistent with increased in vivo HNE activity in these individuals.

Diminished synthesis of an alpha chain mutant, hemoglobin I (alpha-16 lys leads to glu).

In patients heterozygous for abnormal hemoglobins there is usually less than 50% of the mutant hemoglobin present in peripheral blood. The synthetic rates of alpha-chain mutants compared to alpha(A) have not been reported to date. In this study the production of alpha(A)- and alpha(I)-chains has been measured in peripheral blood and bone marrow of two patients with approximately 30% hemoglobin I, an alpha-chain abnormality (alpha(16 lys --> glu)). The results suggest that the decreased amount of alpha(I) compared to alpha(A) is due solely to diminished biosynthesis of the alpha(I)-chains. The relative rates of synthesis of alpha(I)- and alpha(A)-chains are similar in both nucleated red cells and reticulocytes indicating that no change occurs during erythroid cell maturation which preferentially affects either alpha(I) or alpha(A) production.

Adrenocorticotropin, beta-endorphin, and beta-lipotropin in normal thyroid and lung: possible implications for ectopic hormone secretion.

The expression of the proopiomelanocortin (POMC) gene by normal lung and thyroid was examined by measurement of the content of ACTH, beta-lipotropin (beta LPH), and beta-endorphin (beta EP) in porcine lung and thyroid tissue. Acid extracts of normal porcine lung and thyroid tissue each contained appreciable amounts of immunoreactive (ir) ACTH, ir-beta LPH, and ir-beta EP. The content of ir-beta LPH in both tissues exceeded by severalfold, on a molar basis, the content of ir-ACTH and ir-beta EP, suggesting that the common precursor POMC was processed predominantly to peptides other than ir-ACTH and ir-beta EP. A porcine thyroid extract (Calcitare, porcine calcitonin, Armour) showed equivalent levels of beta EP-like immunoreactivity and bioactivity, measured by opiate radioreceptor assay; in contrast, ACTH-like bioactivity, measured by rat zona fasciculata steroidogenesis, was only 4% of ACTH-like immunoreactivity. On reversed phase high performance liquid chromatography, Calcitare showed multiple peaks of ACTH-like immunoreactivity, one of which coeluted with porcine ACTH-(1-39), and two much smaller peaks of beta EP-like immunoreactivity, of which the smaller coeluted with porcine beta EP. These data suggest that both lung and thyroid gland synthesize POMC, which in normal tissue is usually predominantly processed to species other than ACTH and beta EP. Ectopic secretion of ACTH and beta EP by lung and thyroid neoplasms may thus represent the loss of a system(s) normally responsible for processing the precursor beyond ACTH and beta EP.

Phosphorylation sites for ribosomal S6 protein kinases in mouse 3T3 fibroblasts stimulated with platelet-derived growth factor.

Platelet release products and purified platelet-derived growth factor stimulated the phosphorylation of ribosomal protein S6 in cultured mouse Balb/c 3T3 fibroblasts. The post-nuclear fraction of the stimulated cells was enriched in S6 kinase activity specific for sites resembling those phosphorylated within intact cells in response to PDGF as determined by tryptic peptide mapping. 3T3-S6 sites closely resembled those phosphorylated in S6 of rat hepatocytes stimulated with insulin and included sites for both cAMP-dependent and independent kinases.

Further observations on the effects of dietary fatty acid composition on platelet reactivity and blood coagulation in man and the influence of methodology on findings.

Eight human subjects were fed diets enriched in saturated fat (SF), or polyunsaturated fat (PUF) and after each dietary regimen the plasma heparinthrombin clotting time (HTCT) was determined. The HTCT of citrated plasma indicated reduced heparin-neutralizing activity (HNA) after PUF feeding compared with SF feeding. Platelet factor 4 (PF4) levels in the citrated plasma samples demonstrated an inverse correlation with the HTCT (r = 0.62). Experiments with purified PF4 indicated that the PF4 present in citrated plasma could only account for approximately 10% of the HNA. Plasma prepared in a manner which minimized in vitro release of platelet constituents contained significantly less PF4 after PUF feeding and indicated that most of the PF4 found in citrated plasma resulted from in vitro release. The factor Xa inhibitory activity of citrated plasma was not significantly altered by either of the dietary regimens.

A crossreactive antipeptide monoclonal antibody with specificity for lysyl-lysine.

Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgM kappa molecule, with a Kd of 2.8 x 10(-7) M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Surprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly-L-lysine, beta-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I-PDGF-AAL for antibody. 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamic acid [corrected] and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.

Complete covalent structure of human platelet factor 4.

The amino acid sequence of the subunit of human platelet factor 4 has been determined. Human platelet factor 4 consists of identical subunits containing 70 amino acids, each with a molecular weight of 7,756. The molecule contains no methionine, phenylalanine or tryptophan. The proposed amino acid sequence of PF4 is: Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gln-Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser. From consideration of the homology with beta-thromboglobulin, disulphide bonds between residues 10 and 36 and between residues 12 and 52 can be inferred.

Isolation of inhibin from ovine follicular fluid.

Two forms of inhibin with molecular weights of 65,000 and 30,000 (65 and 30 kD) were isolated from ovine follicular fluid using a combination of gel permeation chromatography, reversed-phase high-performance liquid chromatography and preparative polyacrylamide gel electrophoresis. The 65 kD form was partially purified approximately 315-fold whilst the 30 kD form was isolated as two isoforms (29 and 30 kD) of similar biological activity and in greater than 95% purity (1210-fold purification and 4.2% recoveries). On reduction the 30 kD form resolved into four components of 36, 31, 20-21 and 16 kD of which the 20-21 and 16 kD components were similar to the corresponding inhibin subunits isolated from porcine and bovine follicular fluid. The 36 kD component was established as a non-reducible inhibin-like material, based on its binding to antiserum raised against bovine 58 kD inhibin. The nature of the remaining non-reducible 31 kD component is unknown. Two NH2-terminal amino acid sequences (first 13 amino acids) identified in purified 30 kD inhibin were identical to the corresponding subunit amino acid sequences of bovine 31 kD inhibin.

Isolation of inhibin from ovine follicular fluid using reversed-phase liquid chromatography.

This report describes the application of high performance liquid chromatography (HPLC) and associated techniques to the isolation of inhibin from ovine follicular fluid (oFF). Two modes of HPLC have been assessed. High speed gel permeation chromatography on a Waters I-125 support showed that at pH 7.0 inhibin bioactivity was associated with an approx. 80000 mol. wt protein fraction which co-eluted with the bulk of the protein in oFF and gave a yield of 30-44% of the applied activity. Reversed-phase HPLC (RP-HPLC) of a peptide-rich fraction extracted from oFF was also investigated. Using analytical liquid chromatography on octadecylsilylsilica (ODS-silica), two distinct inhibin bioactive fractions were obtained, with a resultant purification of 23-fold and 3-fold respectively and an overall recovery of 8.5%. These results demonstrate that RP-HPLC may be employed as a direct and rapid technique for the isolation of inhibin from oFF.

Isolation of a 31 kDa form of inhibin from bovine follicular fluid.

The introduction of a pH 4.75 precipitation step to a previously described purification procedure from bovine follicular fluid (bFF) resulted in the isolation of a 31 kDa form of inhibin, in addition to 58 kDa inhibin. The procedure was monitored by an in vitro bioassay based on the suppression of the FSH cell content by pituitary cells in culture. The 31 kDa form was purified 5550-fold with approximately 5% recovery. On SDS-polyacrylamide gel electrophoresis a single band was detected with a molecular weight of 31 000 +/- 1500 (mean +/- SD) which upon reduction gave 2 subunits of 20 200 +/- 300 and 14 800 +/- 600. The biological activity expressed on mg protein basis was similar for both 31 kDa and 58 kDa inhibin although on a molar basis the 58 kDa inhibin was 2-3 times higher. A high degree of cross-reaction was observed between both forms in a radioimmunoassay of bovine inhibin using an antiserum raised against the larger form with either iodinated 31 kDa or 58 kDa inhibin as tracer. Based on the subunit composition of the 31 kDa and 58 kDa inhibin, their similar cross-reaction in a radioimmunoassay system and the apparent generation of the 31 kDa inhibin following a pH precipitation step, it is concluded that 31 kDa inhibin is a smaller form of the 58 kDa inhibin resulting from a shortening of the 43 kDa subunit to a 20 kDa subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Posttranslational processing of corticotropin-releasing factor in the ovine tuberoinfundibular system and pituitary.

The present studies were undertaken to characterize the immunoreactive-corticotropin-releasing factor (ir-CRF) in two areas of the ovine tuberoinfundibular system, hypophysial portal blood, and pituitary. With an antiserum raised against synthetic ovine (o)CRF(1-41) and 125I-Tyro-oCRF(1-41) as the tracer, concentrations of ir-CRF (pg/mg wet weight, n = 5) were: paraventricular hypothalamus (PVN), 11.7 +/- 2.5; median eminence (ME), 2276 +/- 296; anterior pituitary (AP), less than 0.5; posterior pituitary (PP), 10.0 +/- 2.2. Analysis of the ir-CRF in these areas on G-75 Sephadex chromatography revealed two main peaks--a 'major' peak which coeluted with synthetic oCRF(1-41) and a 'minor' peak which eluted eight fractions later. These two immunoreactive species of CRF were also found in hypophysial portal blood. When ME extract was analyzed by reverse-phase high performance liquid chromatography (HPLC), the 'minor' peak of ir-CRF eluted before that of CRF(1-41). Since CRF contains Arg35-Lys36 within its sequence, we tested the hypothesis that the 'minor' peak of ir-CRF represented a fragment, or fragments, of the molecule derived by proteolytic cleavage at this site. Tyro-oCRF(34-41) was digested with trypsin and the reaction products were identified by amino acid analysis. Two of these products were CRF(36-41) and CRF(37-41), and both migrated in the 'minor' peak area on G-75 Sephadex chromatography and HPLC. In the CRF(1-41) RIA, serial dilution of both fragments yielded nonparallel displacement curves. However, with 125I-Tyro-oCRF(34-41) as the radiolabeled ligand and Tyro-oCRF(34-41) as the standard, serial dilutions of CRF(1-41), CRF(36-41), and CRF(37-41) generated parallel displacement curves, and the molar cross-reactivities were 90%, 45% and 10% respectively. When the ir-CRF in HPLC fractions of ovine ME was measured in the Tyro-oCRF(34-41) RIA, the molar abundance of the hexapeptide and pentapeptide could be obtained. Calculations based on the premise that the 'minor' peak was solely composed of either the hexapeptide or pentapeptide indicated that CRF(36-41) could account for up to 37% of the total ir-CRF, or that CRF(37-41) could account for up to 73% of the total immunoreactivity. On more discriminating HPLC systems, immunoreactive (ir-) oCRF in the sheep median eminence (ME) could be resolved into five different molecular forms. On three distinct chromatographic systems, four of these immunoreactive species shared retention characteristics identical with synthetic oCRF(37-41), oCRF(36-41), oCRF(16-41) and oCRF(1-41), with the fifth immunoreactive peak as yet unidentified.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro synthesis of low molecular weight proteins in human platelets: absence of labelled release products.

Isolated human blood platelets incubated at 37 degrees C in vitro incorporated labelled amino acids into compounds which included some low molecular weight (less than 80KDa) proteins, as determined by autoradiography after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Non-dialysable plasma factor(s) inhibited both uptake and incorporation, which although unaffected by Actinomycin D, was inhibited partly by Chloramphenicol and almost completely by Puromycin and Cycloheximide, results which confirm that synthesis is directed by pre-existing mRNAs, some of which is mitochondrial. Assuming that the mRNA coding for proteins which are truly "platelet specific" must be present in megakaryocyte cytoplasm, we investigated the possibility that such RNA may be sufficiently stable for its translation to continue in platelets. Although leakage from platelet alpha-granules and cytoplasm during incubation was negligible and platelets retained their secretory potential we were unable to detect radiolabelled proteins in thrombin-released material after incubation. We conclude that either alpha-granule proteins are not synthesised in platelets or their megakaryocyte progenitors, or that their mRNAs become degraded by the time platelets reach the peripheral circulation. Alternatively, the mechanism which concentrates these proteins in granules does not function in circulating platelets.

Acute postprandial lipaemia does not influence the in vivo activity of human platelets.

Platelet function and serum lipid studies were conducted on nine healthy male subjects before and two hours after three separate meals, two of which were rich in fat: saturated fat (SF) or polyunsaturated fat (PUF), and a third control meal which contained no fat. Platelet activity was assessed by determination of in vivo platelet aggregate formation, and plasma levels of the platelet specific proteins platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG). Serum triglyceride levels increased significantly after both fat containing meals but were unaffected by the fat free meal. Serum cholesterol levels were not affected by any of the three meals. Platelet function tests could not detect any alteration of in vivo platelet activity in terms of platelet aggregate formation or plasma concentrations of PF4 and beta-TG. These results are at variance with previously published studies that used less specific assays of platelet activity and which suggested platelet activation shortly after meals rich in SF.